CN108546765B - SNP molecular marker related to Gaoyou duck muscle growth and development traits as well as acquisition method and application thereof - Google Patents
SNP molecular marker related to Gaoyou duck muscle growth and development traits as well as acquisition method and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker related to Gaoyou duck muscle growth and development traits, an acquisition method and application thereof, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1 and is from TNNI1 gene, the 130 th site of the sequence is SNP mutation site, and the base of the site is A or G. The Gaoyou duck bred by the SNP molecular marker has the advantages that the related characters of the weight, the breast and leg muscle weight of the Gaoyou duck are improved, the Gaoyou duck can be used for early selection in family breeding, and molecular basis can be provided in matching. The SNP molecular marker of the invention provides a new method for screening the weight of Gaoyou duck and the weight of breast and leg muscles.
Description
Technical Field
The invention belongs to the technical field of poultry genetic engineering, and particularly relates to an SNP molecular marker related to Gaoyou duck muscle growth and development traits, and an acquisition method and application thereof.
Background
China is the first major country for meat duck production and consumption in the world. The duck meat has low fat content and high unsaturated fatty acid content, and the demand of duck meat products is increased rigidly in the total demand of meat products in China. But the market still has 7-8 hundred million gaps every year, which is far away from the development speed, resource utilization and development scale of the duck industry in China. At present, cherry valley ducks (the average weight of 70 days old is about 3.3kg, the breast muscle rate is 17 percent, and the leg muscle rate is 9 percent) which are introduced from abroad and successfully bred by Beijing ducks have absolute advantages in the domestic meat duck market, south Tech ducks in France also enter the domestic market in recent years, and the excellent local variety resources in China cannot be reasonably utilized and excavated, so that the waste of the excellent local variety resources in China is caused. In meat duck breeding, the selective improvement of the growth and development of skeletal muscles of meat ducks in early growth stage is a barrier for successful breeding and industrial upgrading. With the development of modern biotechnology and information technology, molecular breeding is increasingly applied to improving livestock and poultry varieties to accelerate breeding progress.
The TNNI1 gene belongs to the subunit of Troponin I (TnI). TnI is localized to the striated muscle, and its main function is through Ca2+Binding modulates myosin ATPase activity and thus muscle contraction. The TnI gene contains three subtypes, and is classified into slow skeletal troponin I (TNNI1), fast skeletal troponin I (TNNI2), and cardiac troponin I (TNNI3) according to tissue distribution and expression specificity. TNNI1 gene is specifically expressed mainly in slow-contracting skeletal muscle in human adulthood. Research in pigs shows that TNNI1 has expression in different types of muscle fibers in the porcine embryonic stage, and the expression peak is 120 days in muscles mainly comprising fast white muscle fibers and 60 days in muscles mainly comprising slow muscle fibers; the sheep study shows that the expression level of the TNNI1 gene in muscle gradually increases with the age, the expression level is lowest at half year of age, and the expression level is balanced after 2 years of age. The sheep study shows that the expression level of the TNNI1 gene is related to the diameter, the cross-sectional area and the like of sheep muscle fibers. These reports strongly demonstrate that the TNNI1 gene plays an important role in the development of muscle and growth in animals. Preliminary studies of subject groups showed that TNNI1 mRNA expression was not significantly correlated with diameter and cross-sectional area of duck pectoral and leg muscle fibers. TNNI1 gene may have functions and modes of action in birds different from those in mammals.
The embryonic stage is an important stage of the formation of the muscle of the animal, the number of myofibroblasts is basically fixed, and the number does not increase after birth. The increase in the amount of meat produced after birth is mainly an increase in the diameter and length of the muscle fibers. The pectoral and leg muscles are the major meat-producing parts of ducks. At present, no report about genetic variation of TNNI1 gene and duck muscle growth and development traits is available. The Gaoyou duck is one of three major ducks in China, has fast growth and development and good meat quality, and belongs to a meat and egg dual-purpose local variety. In breeding efforts, it is still easier to change the genetic structure of a variety than to change the genetic characteristics of an individual (artificial mutagenesis). Moreover, on the basis of the existing genes, the genetic structure of the variety is improved by changing the gene frequency, and the potential is also great.
Single Nucleotide Polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genome level. SNP is used as a third-generation molecular marker and has the characteristics of large quantity, wide distribution, strong representativeness, good genetic stability and the like. The method is widely applied to the research of genetic diversity analysis, gene mapping, molecular marker assisted breeding and functional genetics of animals and plants.
Therefore, the research on the SNP molecular markers related to the Gaoyou duck muscle growth and development traits has important significance for the early breeding of Gaoyou ducks.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to Gaoyou duck muscle growth and development traits, an acquisition method and application thereof.
The technical scheme of the invention is as follows:
a SNP molecular marker related to Gaoyou duck muscle growth and development traits is disclosed, wherein a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1 and is from TNNI1 gene, the 130 th site of the sequence is SNP mutation site, and the base of the site is A or G.
The invention also provides a method for obtaining the SNP molecular marker related to the Gaoyou duck muscle growth and development traits, which comprises the following steps:
(1) PCR amplification is carried out on Gaoyou duck genome DNA to obtain a PCR amplification product;
(2) sequencing the PCR amplification product, and judging the genotype through sequence analysis;
(3) and carrying out correlation analysis on different genotypes and Gaoyou duck muscle growth and development traits so as to obtain the SNP molecular marker.
In step (1), the sequence of the forward primer used for PCR amplification is shown as SEQ ID NO. 2, and the sequence of the reverse primer is shown as SEQ ID NO. 3.
In the step (2), the genotype is AA, AG or GG.
In the step (3), the Gaoyou duck muscle growth and development traits comprise body weight, breast muscle weight and leg muscle weight.
The invention also provides application of the SNP molecular marker related to the Gaoyou duck muscle growth and development traits in screening Gaoyou duck weight, breast muscle weight and leg muscle weight traits.
The invention has the beneficial effects that: the invention provides an SNP molecular marker related to Gaoyou duck muscle growth and development traits, which is characterized in that specific primers are designed to amplify a gene fragment containing an SNP site aiming at the SNP polymorphism of a TNNI1 gene, and then sequencing analysis is carried out, so that the single nucleotide polymorphism can be detected simply, quickly, at low cost and accurately. The Gaoyou duck bred by the SNP molecular marker has the advantages that the related characters of the weight, the breast and leg muscle weight of the Gaoyou duck are improved, the Gaoyou duck can be used for early selection in family breeding, and molecular basis can be provided in matching. The SNP molecular marker of the invention provides a new method for screening the weight of Gaoyou duck and the weight of breast and leg muscles.
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FIG. 1 is a graph showing the results of genotyping in the examples of the present invention.
Detailed Description
The present invention will be explained in detail with reference to examples.
The SNP molecular marker related to Gaoyou duck muscle growth and development traits is derived from TNNI1 gene, is positioned at 1397382 th position of TNNI1 gene, has a nucleotide sequence shown as SEQ ID NO. 1, and has a 130 th position as SNP mutation site, wherein the base of the site is A or G.
Aiming at the SNP molecular marker, the invention also discloses an acquisition method thereof, which comprises the following steps:
(1) PCR amplification is carried out on Gaoyou duck genome DNA to obtain a PCR amplification product;
(2) sequencing the PCR amplification product, and judging the genotype through sequence analysis;
(3) and carrying out correlation analysis on different genotypes and Gaoyou duck muscle growth and development traits so as to obtain the SNP molecular marker.
Wherein, in the step (1), the sequence of the forward primer used for PCR amplification is shown as SEQ ID NO. 2, and the sequence of the reverse primer is shown as SEQ ID NO. 3. In the step (2), the genotype is AA, AG or GG. In the step (3), the Gaoyou duck muscle growth and development traits comprise body weight, breast muscle weight and leg muscle weight.
The method for acquiring the SNP molecular marker related to the Gaoyou duck muscle growth and development traits specifically comprises the following steps:
1. collecting duck muscle samples and extracting DNA:
the duck of the invention is from Gaoyou duck group in Jiangsu province, and is a Gaoyou duck group of the same age of the same day fed at the same daily ration level. After the Gaoyou duck was weighed, about 1mL of blood was drawn from the inferior vein of the duck wing using a disposable syringe, injected into a 1.5mL centrifuge tube autoclaved and filled with about 200. mu.l of 2% sterile EDTA (Ethylene diamine tetraacetic acid, EDTA) anticoagulant, shaken gently, recorded the wing number, and stored at-80 ℃ for future use. Meanwhile, after the duck is slaughtered, the lateral heads of the pectoralis major and gastrocnemius muscles on one side are respectively taken down and weighed. Collection of duck blood DNA samples was performed with reference to the instructions of the blood/cell/tissue DNA extraction kit (tiangen, DP 304).
2. PCR amplification of the fragment in which the SNP site is located
Designing a primer containing a site sequence to be detected according to a TNNI1 genome sequence, wherein the nucleotide sequences of the forward primer and the reverse primer are shown as SEQ NO:2-3, the Gaoyou duck genome is used as a template, and the amplification is carried out under the PCR reaction condition under the condition that the forward and reverse primers containing the site sequence to be detected, Taq DNA polymerase, buffer environment and dNTPs exist, and the size of a PCR product is 389 bp. The sequences of the primer pairs are as follows:
the forward primer sequence is: 5'-CTCAGGGTCAGCAGTGTCC-3' (SEQ ID NO: 2);
the reverse primer sequence is as follows: 5'-CTCAGCAGCTACACCGAGG-3' (SEQ ID NO: 3)
The PCR amplification reaction comprises the following specific steps:
1) preparing a PCR amplification reaction system: 50 ng/. mu.l DNA template 1. mu.l, 2mM dNTP 2. mu.l, 3mM Mg2+Mu.l of 0.6. mu.l, 2. mu.l of 10 XPCR reaction buffer, 0.4. mu.l of each of 10. mu.M forward and reverse primers, 0.2. mu.l of 1U/. mu.l of Taq polymerase, and ddH2O to a total volume of 20. mu.l.
2) Setting a PCR amplification reaction program: amplification reactions were performed on a Perkin-Elmer GeneAmp PCR Systems 9600, with the PCR amplification reaction program: denaturation at 95 ℃ for 2min, and extension at 65 ℃ for 10min for 40 cycles (94 ℃ for 90s, 50 ℃ for 1min for 30s, 65 ℃ for 30s) to obtain PCR amplification product.
3) After the reaction, 2. mu.l of the reaction product was electrophoresed in 3.0% agarose gel, 0.5 XTBE, and the reaction was examined for success, and the remaining reaction product was stored at-20 ℃.
3. Genotype determination and correlation analysis
The PCR amplification product is sent to Shanghai bioengineering Co., Ltd for sequencing. And judging the genotype of the locus in the detection population according to the sequencing result. Genotyping results are shown in FIG. 1, where there is only one product peak if the sample is homozygous: an A peak or a G peak. If heterozygous, 2 peaks appear: a peak and G peak.
The different genotypes of the polymorphic site of the TNNI1 gene are subjected to correlation analysis with the weight of ducks, the weight of breast muscles and the weight of leg muscles, the correlation analysis result is shown in Table 1, and the nucleotide sequence shown in SEQ ID NO: the 130 th position on 1 is obviously related to the weight of Gaoyou duck 70 days old, the weight of pectoral muscle and the weight of the head outside gastrocnemius of leg muscle (P < 0.05); the weight, chest muscle weight and leg muscle gastrocnemius lateral head weight of the genotype AA at the age of 70 days are obviously higher than those of the genotypes AG and GG (P is less than 0.05), so that the AA genotype is a dominant genotype. In breeding screens, only AA types are selected, and the genotype can be fixed in the population.
TABLE 1 correlation analysis of different genotypes with Duck weight, Breast muscle weight and leg muscle weight
Note: lower and lower case letters in the same row indicate significant differences (P <0.05) and the same letter indicates insignificant differences (P > 0.05).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and simplifications made in the spirit of the present invention are intended to be included in the scope of the present invention.
Sequence listing
<110> scientific research institute for poultry in Jiangsu province
<120> SNP molecular marker related to Gaoyou duck muscle growth and development traits, and acquisition method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 389
<212> DNA
<213> Duck (Anas platyrhynchos)
<400> 1
ctcagggtca gcagtgtcct gctacacaca ggggcatctt ctgcaaggca ctgactcccc 60
ccctgccctc ctcccgcaga ttaaggacct gaaaatcaaa gtgcttgacc tgcgggggaa 120
gtttaagcga ccgcccctgc ggcgggtccg cgtctccgcc gatgccatgc tgagggctct 180
gctgggctcc aagcacaagg tctccatgga cctcagagcc aacctgaaat ccgtcaagaa 240
ggaggacacg gagaaggtgg gtcgctcccc cagaggcaga gagggctccc gggattgtcc 300
tcgcccttct ccaaggtccc ctgggaagcc tcgagggttt tccaggtacc cagcaggggt 360
actgcagccc cctcggtgta gctgctgag 389
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (unknown)
<400> 2
ctcagggtca gcagtgtcc 19
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (unknown)
<400> 3
ctcagcagct acaccgagg 19
Claims (5)
1. A method for acquiring SNP molecular markers related to Gaoyou duck muscle growth and development traits, which is characterized in that the nucleotide sequence of the molecular markers is shown as SEQ ID NO:1 and is from TNNI1 gene, the 130 th position of the sequence is SNP mutation site, the base of the site is A or G, the method comprises the following steps:
(1) PCR amplification is carried out on Gaoyou duck genome DNA to obtain a PCR amplification product;
(2) sequencing the PCR amplification product, and judging the genotype through sequence analysis;
(3) and carrying out correlation analysis on different genotypes and Gaoyou duck muscle growth and development traits so as to obtain the SNP molecular marker.
2. The method for acquiring the SNP molecular marker related to the Gaoyou duck muscle growth and development traits as claimed in claim 1, wherein in the step (1), the sequence of the forward primer used for PCR amplification is shown as SEQ ID NO. 2, and the sequence of the reverse primer is shown as SEQ ID NO. 3.
3. The method for acquiring the SNP molecular marker related to the Gaoyou duck muscle growth and development trait of claim 1, wherein in the step (2), the genotype is AA, AG or GG.
4. The method for acquiring the SNP molecular marker related to the Gaoyou duck muscle growth and development traits as claimed in claim 1, wherein in the step (3), the Gaoyou duck muscle growth and development traits comprise body weight, breast muscle weight and leg muscle weight.
5. The application of a primer group for detecting SNP molecular markers related to Gaoyou duck muscle growth and development traits in screening Gaoyou duck weight, breast muscle weight and leg muscle weight traits is characterized in that the nucleotide sequence of the molecular markers is shown as SEQ ID NO. 1 and is from TNNI1 gene, the 130 th position of the sequence is SNP mutation site, the base of the site is A or G, the primer group comprises a forward primer and a reverse primer used for PCR amplification, the sequence of the forward primer is shown as SEQ ID NO. 2, and the sequence of the reverse primer is shown as SEQ ID NO. 3.
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| CN112852979B (en) * | 2021-03-25 | 2022-05-10 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to duck breast muscle fiber diameter character and application thereof |
| CN113373240B (en) * | 2021-06-15 | 2024-01-23 | 扬州大学 | Method for identifying gene locus related to duck muscle development |
| CN113388686B (en) * | 2021-07-21 | 2022-11-04 | 江苏省家禽科学研究所 | SNP locus combination for identifying Gaoyou duck varieties and application thereof |
| CN113481305A (en) * | 2021-08-10 | 2021-10-08 | 安徽农业大学 | Molecular marker for identifying duck leg muscle character based on MyoD gene and method and application thereof |
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Non-Patent Citations (2)
| Title |
|---|
| Identification of three novel SNPs and association with carcass traits in porcine TNNI1 and TNNI2;Z. Y. Xu;《Mol Biol Rep 》;20100225;第37卷;第3609–3613页 * |
| Molecular Cloning, Characterization, and Temporal Expression Profile of Troponin I Type 1 (TNNI1) Gene in Skeletal Muscle During Early Development of Gaoyou Duck (Anas Platyrhynchos Domestica);Jingting Shu;《Animal Biotechnology》;20180320;第1-11页 * |
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